1. Field of the Invention
The invention relates generally to methods for screening and obtaining vaccines generated from administration of expression libraries constructed from a pathogen genome. The method further includes identification of one or more antigenic plasmids that will elicit an immune response that is protective against pathogen challenge subsequent to inducing an in vivo immunogenic response. Also included in the invention are particular vaccine compositions protective against Listeria and Mycoplasma.
2. Description of Related Art
While vaccination is one of the most cost-effective medical methods for saving lives, vaccines have not been developed for many of the most serious human diseases, including respiratory syncytial virus (RSV), pneumonia caused by Streptococcus pneumoniae, and diarrhea caused by rotavirus and Shigella. As is evident with the HIV epidemic, the increase in tuberculosis and the endemic spread of malaria and other parasitic diseases, there is an increasing need to develop effective vaccines, yet for many of these pathogens daunting scientific problems have arisen. For example, the influenza virus is notorious for antigenic drift so that new vaccines are constantly being developed; research efforts continue in attempts to identify effective vaccines for rabies (Xiang, et al, 1994), herpes (Rouse, 1995); tuberculosis (Lowrie, et al, 1994); HIV (Coney, et al, 1994) as well as many other diseases of importance in developed and undeveloped countries. Yet there exists no relatively rapid, yet alone systematic, means of identifying an effective vaccine, much less a reasonable assurance that, once identified, the vaccine will be broadly responsive to pathogen challenge.
Many currently used vaccines are composed of live/attenuated pathogens (Ada, 1991) which when inoculated infect cells and elicit a broad immune response in the host. While highly detailed knowledge of the pathobiology is not necessary, at the very least isolation and identification of the pathogen is required. Live vaccines are often superior to antigen or subunit vaccines because of their tendency to elicit a broad level protective response; however, serious disadvantages in using such vaccines include the risk of a vaccine-induced infection, problems with producing and storing the vaccine, and failure to engender any immune response; for example, where antigen presentation is limited. Perhaps the most troubling aspect of using live vaccines is the propensity for actually causing the disease against which protection is intended. Past experience with some of the polio and measles vaccines has demonstrated that this may be a serious risk.
An alternative to the use of live/attenuated pathogen vaccines is to use antibodies to single proteins or to a limited number of proteins associated only with the pathogen. Polyclonal or monoclonal antibodies are readily produced with the aid of modern hybridoma technology, although these techniques are relatively expensive and time consuming. There is also no assurance that antibodies produced in response to an antigen will provide protection against the pathogen providing the antigen; consequently, it may be necessary to test a large number of antigens isolated from a pathogen. Ultimately, no single antigen may prove effective as a vaccine because the ability of subunit or killed vaccine preparations to elicit a broad immune response is generally quite limited.
Certain disadvantages of conventional vaccines are overcome by using what is called "genetic immunization" (Tang, 1992). This technology involves inoculating simple, naked plasmid DNA encoding a pathogen protein into the cells of the host. The pathogen's antigens are produced intracellularly and, depending on the attached targeting signals, can be directed toward major histocompatibility complex (MHC) class I or II presentation (Ulmer, et al, 1993; Wang, et al, 1993). Risk of infection is essentially eliminated and the DNA can be delivered to cells not normally infected by the pathogen. Compared to conventional vaccines, the production of genetic vaccines is straightforward and DNA is considerably more stable than proteinaceous or live/attenuated vaccines. Genetic immunization (a.k.a. DNA, polynucleotide etc. immunization) with specific genes has shown promise in several model systems of pathogenic disease (Davis, et al, 1993; Conry, et al, 1994; Xiang, et al, 1994), and a few natural systems (Cox, et al, 1993; Fynan, et al, 1993). Use of DNA (or RNA) thus overcomes some of the problems encountered when an animal is presented directly with an antigen.
However, despite promising initial results with genetic vaccination, there remains the more basic and unsolved problem of identifying the particular gene or genes of the pathogen that will express an immunogen capable of priming the immune system for rapid and protective response to pathogen challenge. Certain non-viral pathogens and some viruses have very large genomes; for example, protozoa genomes contain up to about 10.sup.8 nucleotides, thus posing an expensive and time-consuming analytical challenge to identify or isolate effective immunogenic antigens. The solution to this problem to date has been to extensively study the pathobiology of the host-pathogen interaction to isolate the protein reacted to by the host during infection. And even with identification of a gene or subunit that elicits a protective immune response, there may still be lacking strong protection because of lack of broad response to the encoded polypeptide.
Significantly, the time and money to identify and develop a vaccine would be greatly reduced if there were available simple, systematic and rapid ways to identify vaccines for specific pathogens without having first to determine at least the fundamental biological properties of the pathogen. Even more important would be vaccines that are broadly effective without any danger of causing the disease against which it is intended to protect.